The Evolution of Pathogen Discovery in Liberia

Lawrence S. Fakoli III

Research Associate

Liberian Institute for Biomedical Research

Website: LIBResearch.org

• Director: Fatorma K. Bolay, PhD.

• In operation since 1975.

• Has been the site of international collaborations studying endemic diseases including but not limited to: • Schistosomiasis

• Malaria

• Ebola virus

• Hepatitis

• Onchosoriasis

• HIV

• Lassa Fever

• Houses the diagnostic capability of the Liberian National Reference Lab

• Website: http://libresearch.org

Liberia Institute for Biomedical Research

• LIBR is operated by the Board of Governors, responsible for all policies affecting the Institute and its activities.

• The Liberian Institute for Biomedical Research (LIBR) was founded 40 years ago and at one time was considered the scientific hub of West Africa.

• Due to a long and brutal fourteen-year war, LIBR was left a shell of its former self with close to no funding or resources.

Liberia Institute for Biomedical Research

• March 2010: NAMRU-3 was requested by AFRICOM to investigate the high incidence of malaria among U.S. Forces

• History of Malaria Cases

• 2003: U.S. Marines 80 / 225

• 2010: 16 cases, 1 death in DEC 2009

• 90% Plasmodium falciparum, 10% P. ovale

• 2010 MID-year GEIS money used to support spray operation and mosquito workshop

• 2011 and 2012 GEIS funded projects

Armed Forces Health Surveillance Center

Objectives: •Implement a country-wide mosquito surveillance. •Map Anopheles spp. distribution using GIS •Support PMI initiatives

Distribution of Anopheles in Liberia

Surveillance and Detection

Distribution of Anopheles in Liberia

Distribution of Anopheles in Liberia

Mosquito

Distribution

Models

Taxonomic

data

Environmental

data

Point occurrence

data

Distribution of Anopheles in Liberia

Distribution of Anopheles in Liberia

Surveillance of Arboviruses Potentially

Involved in Human Febrile Illness in Liberia.

Capability Building

Surveillance of Arboviruses Potentially

Involved in Human Febrile Illness in Liberia.

Capability Building

Surveillance of Arboviruses Potentially

Involved in Human Febrile Illness in Liberia.

05

10152025303540

Average Z-score Max Z-score Positive Cut-Off

Ave.

Z-s

core

Phlebovirus M segment

S-8 Unidentified sandfly (1) Faint ~370 bp Neg. Pos. Neg. Neg. RVFVg Neg.

S-32 Unidentified sandfly (1) Faint ~370 bp Neg. Pos. Neg. Neg. RVFV Neg.

No. Mosquito species (n) PCR result S seg. M seg. L seg. S seg. M seg. L seg.

Surveillance of Arboviruses Potentially

Involved in Human Febrile Illness in Liberia.

Ebola Outbreak Diagnostics.

15

Previous NAMRU-3 laboratory and human capacity

building utilized during Ebola outbreak

Ebola Outbreak Diagnostics.

From conventional PCR to High-throughput

sequencing.

Monitoring of Ebola virus: Liberian counties surveilled.

• Samples derived from EVD patients of seven coastal counties.

• ~1700 positive samples in collection spanning June 2014 to Present.

• ~400 samples met criteria for sequencing by SISPA amplification (<25 Ct).

• 25 nearly complete genomes were published in the initial report in EID in Mar 2015.

LIBR

From conventional PCR to High-throughput

sequencing.

Results from the LIBR Sequencing Center: Ebola virus monitoring.

From conventional PCR to High-throughput

sequencing.

Monitoring of Ebola virus: Viral Evolution

• Introduction events – Liberian outbreak most likely occurred from a single introduction of a clade 2 virus.

• Mutation rates agree with those published by other groups: ~9.17 x 10

• Possible introduction from Liberia into Mali.

4

From conventional PCR to High-throughput

sequencing.

Monitoring of Ebola virus: Target Erosion

• Minimal target erosion observed in Diagnostics used at LIBR.

• More substantial changes seen in publicly available Zaire Ebola virus probes but not all are in use.

• Minimal target erosion seen in therapeutics since the original report in mBio, 2015.

• Risk assessment for efficacy: Low.

From conventional PCR to High-throughput

sequencing.

Monitoring of Ebola virus : Complete, timely, sequence characterization

• Sample 1413 was the first sample completely prepared and sequenced by Liberian staff.

• Reports to the MOH, CDC, WHO and DOD were made available within 7 days of sample receipt for the February samples.

• The public report was made publicly available Apr 22, 2015.

From conventional PCR to High-throughput

sequencing.

Results from the LIBR Sequencing Center: Molecular Evidence of Transmission

From conventional PCR to High-throughput

sequencing.

Transmission Molecular Evidence : Possible Sexual Transmission

• Patient 1, contracted and died of Ebola virus 28 days after the last known positive case in Liberia in March of 2015.

• Case history of the known contacts of Survivor A.

• The viral genome did not match any of the most recent sequences.

• The only known contact determined from the epidemiological study was unprotected sexual intercourse with Survivor A. Declared convalesced (Ebola negative) in September of 2014.

Christie et al, MWWR, 2015.

From conventional PCR to High-throughput

sequencing.

Transmission Molecular Evidence : SNP Evidence

• SPB Cluster follows a single introduction event and was the last circulating lineage known in Liberia.

• Low titers made sequence recovery difficult, however, signature recovered matches Survivor A.

• Other family members sequenced match the SPB cluster.

• Resulted in a revision of the convalescent sexual contact recommendations by CDC and WHO.

Case Relationship 4107 8592 16636 4384 12996 18405 6056 16514

MH13203 Patient 1 A T A C A A C A

MH2012684 Survivor A N T A N N N N N

CDC659=0 CN1 Surv. Brother G N G C N N C A

CDC/NIH-1469 Surv. Ex Wife G A G N C N A G

LIBR0993 SPB (Jan 20, 2015) G A G A C - A G

LIBR1195 SPB (Feb 2, 2015) G A G A C - A G

LIBR1413 SPB (Feb 14, 2015) G A G A C - A G

From conventional PCR to High-throughput

sequencing.

• Naval Medical Research Unit No. 3 (NAMRU-3)

• United States Army Medical Research Institute for Infectious Diseases (USAMRIID)

• Armed Forces of Liberia

• National Reference Laboratory

• Liberian Ministry of Health; National Diagnostic Unit (NDU)

• National Malaria Control Program (NMCP)

• Center for Disease Control and Prevention (CDC)

• U.S. Agency for International Development (USAID)

• Navy Entomology Center of Excellence (NECE)

We would like to thank our collaborators!